PROJECT SUMMARY We have identified a subset of CD8+ cells programmed to inhibit activation and expansion of helper T (TH) cells through recognition of the class Ib MHC molecule Qa-1 (HLA-E in man). Mice that express a Qa-1 point mutation that disrupts binding of Qa-1 to the TCR/CD8 co-receptor of Treg develop dysregulated TFH responses and die of systemic autoimmune disease 9-12m after birth. Regulatory activity is mediated by <5% of CD8 T cells that express a diagnostic triad of surface receptors. We have also shown that the Helios transcription factor (TF) stabilizes the CD8 Treg genetic program and have recently identified the highly restricted T-cell receptor (TCR)?? repertoire that may mediate Ag-specific recognition by CD8 Treg. We use these findings to trace the development of this regulatory CD8 lineage and apply these insights towards development of novel therapeutic approaches to autoimmune disease. We propose here to test the premise that Qa-1-restricted CD8 Treg represent a unique regulatory lineage of CD8 cells that express a Helios-dependent genetic program and a restricted set of TCR specific for Qa-1/HA-E-associated self-peptides. In Aim 1, we will define the contribution of the TCR to intrathymic CD8 Treg selection and differentiation using Ag-specific TCR knock-in (KI) and retrogenic mice that express a TCR specific for Qa-1-restricted self-peptides. This analysis will also allow dissection of the molecular requirements for thymic selection and differentiation of class Ib MHC-dependent CD8+ Treg. Single-cell transcriptome analysis will be used to define the relationship between TCR specificity for Qa-1?peptide ligands and shaping of the lineage-specific genetic program of Ag-specific CD8 Treg. In Aim 2, we will define the contribution of the Helios TF to thymic and post-thymic differentiation of CD8 Treg. This approach will entail measurement of the impact of specific deletion of Helios at defined stages of Ag-specific CD8 Treg differentiation. The results from these studies and SA1 will provide a foundation for investigation of the interaction between Ag-specific CD8 Treg and target cells in disease settings (Aim 3). Here we will define the inhibitory interaction between Ag-specific CD8+ Treg and its target cells. Since Helios-dependent expression of NKG2D costimulatory receptors may be essential to CD8 Treg signaling, we will characterize the contribution of this costimulatory receptor to CD8 Treg activation by stress-associated Qa-1?Hsp60 and NKG2D ligands (NKG2D- L). Insight into this interaction will be applied to the design of nanoparticle-based therapeutic approaches to autoimmune disease.